Fluorescence in situ hybridization with cosmid clones for the detection of human cytomegalovirus DNA in peripheral blood leukocytes

 Radioactive in situ hybridization techniques or enzymatic detection procedures of hapten-modified human cytomegalovirus (HCMV) probes have been widely used for studying the infection of peripheral blood leukocytes with HCMV. This report describes significant improvements in terms of signal resolution which can be obtained by applying a highly sensitive fluorescence in situ hybridization (FISH) technique in conjunction with a large subgenomic HCMV DNA probe. Three cosmid clones spanning 119.1 kb of the HCMV genome (230 kb) were used to construct the digoxigenin-11-dUTP-labeled probe which was found to be superior to a total HCMV probe representing the entire genome. Crucial hybridization parameters were analyzed systematically in order to ensure optimal resolution power and sensitivity. The protocol was successfully applied to HCMV-infected fibroblasts and peripheral blood leukocytes of 12 transplant patients and unambiguously facilitated the precise intracellular localization of HCMV genomes in infected cells. Because of its excellent resolution properties, accompanied by the virtual absence by any background staining, we recommend the use of this protocol as a sensitive approach for further virological analyses of the interactions between HCMV and peripheral blood leukocytes at the single-cell level. Accepted: 16 February 1996     

The dominant phosphoprotein pp65 (UL83) of human cytomegalovirus is dispensable for growth in cell culture.

The phosphoprotein pp65 (ppUL83) of human cytomegalovirus (HCMV) is abundantly synthesized during lytic infection in cultured fibroblasts. As a major constituent of extracellular particles, it gains entry to infected cells immediately after adsorption and subsequently translocates to the cell nucleus. This efficient transport is mediated by unique nuclear localization signals. To study the function of pp65, a viral deletion mutant was constructed by replacing the pp65 gene with the bacterial neomycin phosphotransferase gene, driven by the simian virus 40 early promoter. The resulting virus, RVAd65, could be grown and selected on human fibroblasts without complementation. The deletion of the pp65 gene in RVAd65 was verified by using Southern blot and PCR analyses. The lack of expression from the gene was investigated by immunoblotting with pp65-specific monoclonal antibodies. Single-cycle growth analyses showed that RVAd65 grew to levels of infectivity comparable to those of the wild-type virus. Therefore, pp65 is nonessential for the growth of HCMV in human fibroblasts. Electron microscopy revealed no differences in the processes of virion morphogenesis, although the maturation appeared to be delayed. However, the kinetics of expression of the immediate-early genes UL122 and UL123, the early gene UL44, and the late gene UL32 were the same in RVAd65-infected cells as in wild-type virus-infected cells in immunoblot analyses. In vitro phosphorylation assays showed that some of the virion proteins were labelled to a markedly reduced extent by virion-associated kinases in RVAd65 compared with wild-type virus. We therefore conclude that although deletion of the pp65 gene does not abolish replication of HCMV, a recombinant virus lacking pp65 displays phenotypic alterations compared with wild-type virus during growth in cultured fibroblasts.     

DNA rearrangements in Euplotes crassus coincide with discrete periods of DNA replication during the polytene chromosome stage of macronuclear development.

Macronuclear development in Euplotes crassus begins with polytenization of micronuclear chromosomes and is accompanied by highly precise excision of DNA sequences known as internal eliminated sequences and transposon-like elements (Tecs). Quantitation of radiolabeled-precursor incorporation into DNA indicates that DNA synthesis during formation of polytene chromosomes is not continuous and occurs during two distinct periods. We demonstrate that the timing of Tec excision coincides with these replication periods and that excision can occur during both periods even at a single locus. We also show that Tec and internal eliminated sequence excisions are coincident in the second replication period, thus providing further evidence for similarity in their excision mechanism. Inhibition of DNA synthesis with hydroxyurea diminishes Tec element excision, indicating that replication is an important aspect of the excision process.     

Testosterone-regulated expression of enzymes involved in steroid and aromatic hydrocarbon catabolism in Comamonas testosteroni.

The effect of testosterone as the sole carbon source on protein expression was analyzed in Comamonas testosteroni. Testosterone simultaneously induced the expression of steroid- and aromatic hydrocarbon-catabolizing enzymes and repressed one amino acid-degrading enzyme. It is suggested that steroids play a regulative role in catabolic enzyme synthesis during adaptive growth of C. testosteroni.     

The influence of cadmium on the biliary excretion of eosine and bromsulphthalein and on microsomal monooxygenase activities in the rat

Cadmium sulfate x 8/3 H2O (0.3, 0.6, 1.5 and 2.0 mg/kg) administered simultaneously with eosine and bromsulphthalein (120 mumol/kg i.v.) did not significantly change the biliary excretion of the dyes. After a 3 days pretreatment with 2.0 mg/kg CdSO4 i.p. a body weight loss and an increase in the relative liver weight when calculated on body weight were observed together with an enhanced bile flow, but the excretion of the dyes was not markedly influenced. Ethylmorphine N-demethylation and ethoxycoumarin O-deethylation activities as well as microsomal cytochrome P-450 concentration were diminished by about 50%. It can be concluded that in male rats the hepatic microsomal monooxygenase system is more sensitive towards cadmium than the hepatic transport system for organic anions.